2.11. Immunoblotting of Nuclear Factor E2-Related Factor 2 (Nrf-2)

Equal amounts of protein (30 µg) from treated and untreated Caco2 cells were electrophoresed through an SDS-polyacrylamide gel (10% resolving gel) in Tris-glycine buffer at 90 V for 2 h. The proteins were transferred from the SDS-polyacrylamide gel to Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA) using a wet transfer system. Membranes were developed using WesternBreeze Chromogenic Anti-Rabbit Kit (WB7105, Invitrogen, Carlsbad, CA, USA) and rabbit polyclonal primary anti-Nrf-2 antibody (sc-722, Santa Cruz Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. The protein bands were detected by staining with the BCIP/NBT substrate and visualized with the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). Quantification of bands on Western images was done using the Image Lab software (ver. 5.2, Bio-Rad, Hercules, CA, USA). To normalize Nrf-2 levels, Nrf-2 band intensity was divided by β-actin (A1978, Sigma-Aldrich, St. Louis, MO, USA) band intensity (which serves as reference protein), and represented as percentages of control.